title: The C. reinhardtii CF1 with the mutation betaT168S has high ATPase activity.
abstract: We have generated the mutation T168S in the beta subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF1 and the alpha3beta3gamma complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF1 exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The alpha3beta3gamma complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+. Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.
authors.affiliation: The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Hu
authors.forename: H
authors.initials: H
authors.lastname: Strotmann
authors.forename: N
authors.initials: N
authors.lastname: Shavit
authors.forename: S
authors.initials: S
authors.lastname: Leu
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000254:growth & development; D002734:Chlorophyll / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002734:Chlorophyll; D000431:Ethanol; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases